Analytical work station

ABSTRACT

This invention encompasses a work station for conducting assays which comprises filtering assembly, a slide reading assembly, and a slide with a porous filter membrane wherein the slide has a means for aligning the slide within the filter assembly to filter reagents on predetermined portions of the porous filter membrane and also to position the slide in the slide reading assembly so that detection will occur in those same predetermined locations.

RELATED APPLICATIONS

This is a division of application Ser. No. 07/033,169, filed Mar. 16,1993, now abandoned, which is a division of application Ser. No.07/297,767 filed Jan. 17, 1989, now U.S. Pat. No. 5,252,293.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of analytical devices where analytesare filtered and bound to a membrane and the membrane is then analyzedfor the presence of analyte.

2. Description of the Prior Art

The prior art describes a wide variety of filtering devices ranging fromfilter paper to individual test units which involve binding specificbinding substances such as antibodies and antigens to porous filtermembranes as described in U.S. Pat. No. 4,642,285. U.S. Pat. Nos.4,703,353; 4,591,550; 4,758,786 and 4,704,353 and references thereindisclose a photoresponsive electrode generally adaptable to measurementsmade in the present invention. U.S. Ser. No. 065,418 filed Jun. 18, 1987assigned to the same assignee as this application discloses a ZeroVolume Electrochemical Cell where analytes on porous filter membranesare measured by means of a photoresponsive electrode. U.S. Ser. No.258,894 assigned to the same assignee as this invention, is directed toHapten Derivatized Capture Membranes useful as membranes for the slidesof this invention.

References of interest include U.S. Pat. Nos. 4,020,830 to Jense, etal.; 3,975,238 to Bean, et al.; 4,238,757 to Schenck; 4,486,272 toFujihira, 4,293,310 to Weber; and 4,444,892 to Malmros; andInternational Patent Publications Nos. WO83/02669 and WO85/04018. Seealso "Experimental Electrochemistry for Electrochemists," Sawyer andRoberts, Wiley-Interscience, pp. 350-353.

U.S. patents of interest also include U.S. Pat. Nos. 4,168,146, whichconcerns a test strip for immunoassays, where the extend to which ananalyte travels is related to the amount of analyte in the medium;4,298,688, which involves a three-zone strip, where the extent of travelof an enzymatic product is determinative of the amount of glucoseanalyte; 4,299,916, which concerns an assay technique employing asupport for detection of the analyte; 4,361,537, which employs strips inconjunction with RIAs; 4,366,241, which concerns employing a small testzone for concentrating a particular component of the assay medium in asmall area; 4,435,504, which concerns an immunochromatograph employingchanneling; 4,442,204, which concerns using homogeneous assay reagentson solid support where displacement of labeled conjugate-analyte complexby analyte provides the desired signal; 4,533,629, which employs asimultaneous calibration technique for heterogenous immunoassays;4,446,232, which employs a solid support having a zone occupied bylabeled conjugate, followed by receptor, where binding of analyte to thelabeled conjugate allows the labeled conjugate to traverse the receptorzone to a detection zone; 4,447,526, which employs a homogenous specificbonding assay system in conjunction with carrier matrix; and 4,454,094,which involves displaced apart layers through which a medium traverses,where reagent from one layer diffuses to the other layer in relation tothe amount of analyte in the medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Perspective view of the analytical work station.

FIG. 2 Top view of filter assembly.

FIG. 3 Cross sectional 3-1 view of FIG. 2.

FIG. 4 Disassembled filter assembly.

FIG. 4a Top view of the slide.

FIG. 5 Schematic of the vacuum system.

FIG. 6 Top perspective view of the slide reading assembly.

FIG. 6a Internal perspective view of the slide reading assembly.

FIG. 7 Cross-section of the filter assembly.

FIG. 7a Holes in inner wall section of housing.

FIG. 8 Schematic diagram of circuit.

FIG. 9 Alternating current response curve.

FIG. 9a First derivative of the curve of FIG. 9.

FIG. 10 Standard curve for DNA determination.

SUMMARY OF THE INVENTION

This invention encompasses a work station for conducting assays whichcomprise a filtering assembly, a slide reading assembly, and a slidewith a porous filter membrane wherein the slide has a means for aligningthe slide within the filter assembly to filter an analyte or analytecomplex at a plurality of predetermined locations on the porous filtermembrane and also to position the slide in the slide reading assembly sothat detection will occur in those same predetermined locations.

This invention encompasses an analytical work station which has afiltering assembly, a slide with a porous filter membrane and a slidereading assembly.

The filtering assembly comprises a combination of:

1. a block with a top and bottom surface, the block having a pluralityof channels with inlet ports on the top surface and exit ports on thebottom surface and a first alignment means;

2. a vacuum plate having an upper and lower surface wherein the lowersurface has a vacuum fitting defining an area on the lower surface, witha plurality of vacuum ports from the upper to lower surface within thearea defined by the vacuum fitting, the vacuum plate having a secondalignment means;

3. a slide defining an opening which is covered by a porous filtermembrane and further having a third alignment means.

The block and the vacuum plate fit together to form a space forremovably inserting the slide between the bottom surface of the blockand the upper surface of the vacuum plate and the first, second, andthird alignment means align the block, vacuum plate, and slide such thatthe exit ports of the block, porous filter membrane, and vacuum portsare aligned so that when a vacuum is applied through the vacuum fitting,liquids in the block channels separately flow through the exit ports,through the porous filter membrane, and through the vacuum ports so thatliquids from one channel do not mix with liquids from other channels onthe porous filter membrane. The analytes or analyte complexes in thefiltered liquid are fixed on the porous filter membrane in apredetermined location so as to substantially concentrate the analyte orcomplexes of the analyte.

The slide is a thin flat generally rectangular plastic sheet with afront and rear end. The front end of the slide has a notch as a meansfor aligning the slide both in a filter assembly and a reading assembly.The rear end of the slide has an area for manually grasping the slide.The front, approximately a third of the slide, defines an opening whichis covered with a porous filter membrane. A section of the front onethird of the slide is narrower than the rear section of the slide. Thenarrower section also serves as a means for aligning the slide in both afiltering assembly and the slide reading assembly. The alignment meansprovide for filtration of analyte onto predetermined locations on theporous filter membrane in the filter assembly and for detection ofanalyte at these same predetermined locations in the slide readingassembly. The porous filter membrane has a specific binding substancesuch as a hapten, for example, biotin so that the porous filter membranecan capture a specifically binding analyte complex at a specificlocation on the filter membrane. For example, an avidin or streptavidincomplex is used in conjunction with a biotin labeled membrane.

The slide reading assembly has a housing with a top, bottom, and innerand outer sidewall sections. The inner side wall section has at leastone window or a plurality of holes as windows with a photoresponsiveelectrode mounted above the windows on the inside of the inner wallsection so that light from an external source can pass through the holesto the external surface photoresponsive electrode.

The top section of the housing has an opening for receiving a slide tobe analyzed. The slide has a porous filter membrane with analyte oranalyte complexes to be determined at predetermined locations on theporous filter. The housing has a means for aligning, theanalyte-containing region of the porous filter membrane on the internalsurface of the photoresponsive electrode opposite the holes in thehousing which allow light to pass to the photoresponsive electrode.

A resilient plunger is mounted in the outer side wall section of thehousing opposite the photoresponsive electrode. The plunger has a stemand pad which is pivotally mounted on the stem and located within thehousing such that when the plunger stem is externally pushed into thehousing by a stepping motor the pad compresses the porous membraneagainst the inner surface of the photoresponsive electrode. This reduceselectrolyte volume and improves sensitivity.

There is a liquid chamber within the housing with exit and entranceports for liquids located in the top section of the housing. The innersurface of the photoresponsive electrode is within the liquid chamberand exposed to the liquid. The porous filter membrane is aligned abovethe inner surface of the photoresponsive electrode also in the liquidchamber.

There is a light source comprising an array of light emitting diodes(LEDs) for independently irradiating portions of the photoresponsiveelectrodes with intensity modulated light through the holes in thehousing. The area of the photoresponsive electrode exposed to intensitymodulated light is opposite the area of the porous filter membranehaving filtered analyte or analyte complex.

There is also an electrical means for measuring the rate of surfacepotential change of those portions of the photoresponsive electrodewhich are exposed to the intensity-modulated beam of light through theholes in the housing. A chemical reaction which alters the surfacepotential is occurring between the analyte or analyte complex on theporous filter membrane and a reagent in the liquid in the liquid chamberso as to change the surface potential of an exposed portion of thephotoresponsive electrode. The electrical means includes a counterelectrode or reference electrode and a control electrode, an operationamplifier, and a computer for processing the signal data.

Thus, in operation a reaction between specific binding substances isconducted in a test tube, for example, the reaction between DNA, enzymelabeled anti-DNA, SS-binding protein bound to a biotin, and avidin orstreptavidin. This complex is filtered through a biotinylated porousfilter membrane such that the (enzyme containing) analyte complex iscaptured by the porous filter membrane in small spots at a predeterminedlocation on the membrane. The locations of these spots are fixed by thealignment means in the block, slide, and vacuum plate. The slide isremoved from the filter assembly and placed in the slide readingassembly which contains a substrate to the enzyme in the liquid in theliquid chamber. The plunger is pushed in by a stepping motor to pressthe pad of the plunger against a photoresponsive electrode. Thisdecrease in volume increases sensitivity by decreasing the amount of pHbuffer in the measurement volume. The alignment means in the readingassembly aligns the enzyme-containing spots of the porous filtermembrane opposite the holes for light in the housing. The reaction ofenzyme and enzyme substrate results in a change in surface potential inthe local area of the photoresponsive electrode which is measured by theabove electrical means in conjunction with the action of theintensity-modulated light on the photosensitive electrode.

Thus, the filter assembly provides for concentrating a complexcontaining the analyte to be determined in a small area of a porousfilter membrane which is predetermined by the alignment means. Thispredetermined location of the spots on the filter membrane can then bealigned with a specific reading location in the slide reading assemblyby complimentary alignment means. The pad which compresses the porousfilter membrane against the photoresponsive electrode uniformly reducesvolume and increase sensitivity. In this way picogram (pg) quantities ofanalyte such as DNA can readily be determined. The filter and slidereading assemblies provide for filtering and reading of standards,controls and unknowns under similar conditions as well as for the easeof identification of the sample, and for obtaining an analytical resultfrom the same sample.

DETAILED DESCRIPTION OF THE INVENTION The Slide

FIG. 4a is a top view of the slide.

The slide 4a is made of a thin plastic sheet and has an opening which iscovered with a porous filter membrane 5. The notch 3 at the front end ofthe slide and the angle 25 defined by the narrow and wide portion of theslide serve as means for aligning the slide in the filter assembly andthe slide reading assembly. Slits 3a permit the notch to resilientlyexpand. The rear end of the slide has an area 7 for manually graspingthe slide. A porous filter membrane suitable for practicing thisinvention is described in great detail in Ser. No. 258,894 assigned tothe same assignee of this application. Ser. No. 258,894 is incorporatedherein by reference.

The porous filter membrane is capable of filtering from a solutioncontaining a specifically binding complex having an anti-hapten bound toa binding member of the specifically binding complex.

The material of the porous filter membrane is selected from material towhich protein or other macromolecule can be adhered. A variety ofmaterials may be used. Those skilled in the art will appreciate thatporous membranes made of nylon, cellulose acetate, polyolefin,polyacrylamide, nitrocellulose or other porous materials may be employedin the present invention. Other synthetic or naturally occurringmaterials which will adhere a protein or other macromolecule may also beused. A preferred membrane is made from nitrocellulose. Physicalcharacteristics of the porous filter membrane in the slide are:

(a) pore size:0.25 to 12.0 μm

(b) thickness:50 to 180 μm

(c) bubble point:75 to 6 psi

A membrane having 1-10 m mol (per liter of compressed membrane volume)buffering capacity is preferred.

Haptens are substances which do not elicit antibody formation unlesscomplexed to macromolecules and which may be employed as specificorganic materials for which specific binding substances can be provided.Antibodies to haptens can be formed by binding the hapten to a proteinso as to elicit an antibody response. A specific binding substance isany substance or group of substances having a specific binding affinityfor the hapten to the exclusion of other substances. The hapten must beable to bind to a protein or other macro-molecule directly or through anextended linking group. Examples of haptens which may be used includesteroids such as estrone, estradiol, testosterone, pregnanediol andprogesterone; vitamins such as B₁₂, biotin and folic acid;triiodothyronine, thyroxine, histamine, serotonin, digoxin,prostaglandins, adrenalin, noradrenalin, morphine, vegetable hormonesand antibiotics such as penicillin.

When the hapten is a substance having a naturally occurring receptor,the receptor can be utilized as the anti-hapten provided the receptorcan be isolated in a form specific for the hapten. Illustrative haptenswhich have naturally occurring receptors include thyroxine, manysteroids, polypeptides, such as insulin, angiotensin, biotin and manyothers. Receptors for this class of haptens are usually proteins ornucleic acids.

Extended linking groups are groups that will bind the hapten to theprotein or macro-molecule in such a way that the hapten has betteraccess to the anti-hapten. Where an extended linking group is notneeded, a hapten, such as biotin without an extended linking group, isbound to a functional groups on a membrane or to a function group on aprotein which can be disbursed on the membrane.

Preferentially the extended linking group having an hapten bound to oneend will be bound to the protein or macro-molecule with an amide bond;the amine of the amide bond arising from the protein and the carboxyl ofthe amide bond arising from the carboxy terminus of the extender group.Free carboxyl or hydroxyl groups on proteins can likewise be used.

The proteins used as carriers include, but are not limited to, bovinserum albumin (BSA), bovine gamma globulin, and fibrinogen. A preferredmembrane for use with the slide has 5-20 molecules of biotin bound tobovine serum albumin (BSA). The BSA is further adhered to the surface ofthe membrane. The preferred linking group is the following: ##STR1##

Complexes typically removed from solution by the membrane include:

    ______________________________________                                        Porous Filter Membrane                                                                       Complex                                                        ______________________________________                                        membrane - hapten                                                                            anti hapten-Ab Ag-Ab (enzyme)                                  membrane - (biotin)                                                                          (streptavidin)-Ab' Ag' Ab (enzyme)                             ______________________________________                                    

The antibodies employed may be either polyclonal or monoclonalantibodies and are produced in response to the target antigen of theassay. Methods for the production of antibodies to various biologicalsubstances are well known in the art.

The antigens targeted by the assay include, but are not limited toantigens such as IgE, prostatic acid phosphatase, prostate specificantigen, alphafetoprotein, carcinoembryonic antigen, leutenizinghormone, creatine kinase MB, Human Chorionic Gonadotropin (HCG) andother antigens in serum, plasma, urine, or other liquid media.

Polydeoxyribonucleotides can be determined by reactions with singlestrand DNA binding protein (SSB) and anti-DNA antibodies. Thus, variouscombinations of labeled SSB or anti-DNA and biotinylated SSB or anti-DNAare employed. In this embodiment streptavidin is bound to thebiotinylated SSB or anti-DNA so that the complex can be bound to thecapture membrane having biotin. The article in Biochemistry, 25:21(1986) describes the large scale over production of single-strandbinding protein (SSB) from E. coli. Monoclonal antibodies to DNA havebeen used to measure DNA in biological fluids, Journal of ImmunologicalMethods, 88, (1986) 185-192.

This complex and membrane can be viewed as follows:

    ______________________________________                                        Porous Filter Membrane                                                                       Complex                                                        ______________________________________                                        membrane - biotin                                                                            streptavidin-biotin-anti-DNA/DNA-                                             SSB-enzyme                                                     ______________________________________                                    

Examples of enzymes which may conveniently be employed are, malatedehydrogenase, lipase, delta-5-ketosteroid isomerase, yeast alcoholdehydrogenase, yeast glucose-6-phosphate dehydrogenase, alphaglycerophosphate dehydrogenase, triose phosphate isomerase, horseradishperoxidase, alkaline phosphatase, asparaginase, glucose oxidase,beta-galactosidase, and more preferably, urease. Normally, it ispreferred that the enzyme be in a pure form, free of contaminatingproteins.

The preparation of the enzyme-labelled biological substances can beaccomplished in various ways known in the art. Examples of the couplingof biological substances to enzymes are described in, for example, L. A.Steinberger, Immunocytochemistry, Prentice Hall, N.J. (1974).

The enzyme label on porous filter membrane is made to react with anenzyme substrate and the extent of reaction is measured by thephotoresponsive electrodes and methods described in U.S. Pat. Nos.4,591,550, 4,704,353, and U.S. patent application Ser. No. 876,925,filed Jun. 20, 1986 and assigned to the same assignee as thisapplication. The slide reading assembly is an adaptin and improvement ofthese devices to read the predetermined locations on the membrane wherethe enzyme has been filtered.

The photoresponsive electrode can be influenced by a wide variety ofredox systems where the redox reaction occurs at the surface of thephotoresponsive electrode where the light strikes the surface of thephotoresponsive electrode.

The Filter Assembly

FIG. 1 is a perspective view of the analytical work station 1. In thisembodiment there are four filter assemblies 2 and a slide readingassembly 40. FIG. 2 is a top view of a filter assembly which shows afilter block 10, vacuum plate 11 and slide 4 and FIG. 3 is across-sectional view of 3--3 of FIG. 2. The filter block 10 has aplurality of channels 12 which narrow from the inlet port 13 to the exitport 14. The exit ports 14 are centrally located in a smooth surfacearea 14a at the bottom end of the block. The bottom surface of the blockhas a hole 23 as an alignment means to align the slide with the vacuumplate 11. The block has small projections 15 at the lower end of theside walls which are gripped by the clamping mechanism spring clip 17through opening 16 to fix and release the slide between the filter block10 and the vacuum plate 11 by turning lever 18 as shown in FIGS. 1 and3.

The vacuum plate 11 has on its upper surface a plurality of radialcylinders 19 through which pass the vacuum ports 20 as shown in FIG. 3which is a cross-section of 3--3 of FIG. 2, also see FIG. 4. The smoothlower surface of the filter block and the smooth upper surface of theraised cylinders of the vacuum plate compress the membrane 5. When thelever 18 is turned the rotation of the lever moves the spring block 29up and down as the cam 28 mounted on the lever shaft rotates 90 degrees.The spring clips 17 follow a cam surface 27a and spread in the upperposition allowing an easy removal of the filter assembly from thevacuum. In the lower position the spring clips are closed firmly andwithout any side forces hold the filter block locked against the vacuumplate. This compression prevents fluid from one filter pathway 21crossing over to another filter pathway 21a. The vacuum plate has a peg22 which interacts with the hole 23 of the block and the notch 3 of theslide (shown in FIG. 4a) to align the exit ports 14 in the block and thevacuum ports 20 in the vacuum plate. Slits 3a provide for expanding thenotch 3 to resiliently clamp the peg 22. The vacuum plate further has adepression 24 which fits the shape of the slide 25 to rigidly fix theslide in place as shown in FIG. 2. Turning 18 in FIGS. 3 and 4 releasesthe pressure exerted by block 10, and thus permits the slide 4 to beremoved from the filtering assembly.

FIG. 4 shows the filter assembly disassembled into its component partsand further illustrates the arrangement of the block 10, manifold bottom8, slide 4, and vacuum plate 11 as they sit in the vacuum manifold 9.Further illustrated in FIG. 4 are the liquid level sensor 26, manifoldinsert 27, and check valve 27b which engages vacuum fitting 27c on thevacuum plate 11. FIG. 4 further illustrates how lever 18 interacts withcam 28 and spring block 29 to spring clip 17 which has hole 16 engagedwith projection 15 of the filter block 10. Thus turning 18 lowers theblock 10 and releases the porous filter membrane 5.

FIG. 3 illustrates the interaction of clamping mechanism 18, 28, 29, 17,16 and further shows the engagement of spring clip 17 with the manifoldinsert 27 at point 27a. The manifold insert 27 engages the vacuum plate11 at the vacuum fitting 27c. The vacuum ports 20 lie within the vacuumfitting 27c and the check valve 27b lies within the manifold insert 27.Projections 27d push against the check valve 27b to open the checkvalve.

The vacuum system is schematically illustrated in FIG. 5. The vacuumpump 30 is controlled by the vacuum pump control 31. The vacuum to thevacuum manifold 32 is controlled by the vacuum control 33 which operatesa proportional valve 34. A pressure transducer 35 provides for thevacuum sensor output 36.

The vacuum manifold serves as an effluent reservoir and as a vacuumreservoir. The individual filter assembly receptacle 9 in the manifoldhas a manifold insert with a check valve 27b, which is activated withthe insertion of the filter base to start the vacuum flow. The liquidlevel sensor 26 determines when the manifold should be emptied of wastefluid.

The vacuum system provides vacuum to pull solutions in the channels ofthe filter block through the porous filter membrane on the slide by wayof the vacuum ports.

The Slide Reading Assembly

The slide reading assembly is illustrated in FIGS. 6, 6a, 7 and 7a. Thehousing 40 has a slow 41 in the top end 42 for inserting the slide 4.The housing has an area with a plurality of holes 43 or windows in theinner wall section 44 (see FIG. 7a) and a photoresponsive electrode 45mounted over the holes. This photoresponsive electrode 45 has atransparent layer of silicon dioxide which defines areas of transparentcircular spots 46 which are aligned with the holes 43 in the housing.The windows 43 in the housing and transparent circular spots 46 permitlight to pass from the array of LED's 47 from outside the housing to thesurface of the photoresponsive electrode as shown in FIGS. 7 and 7a.Within the reading assembly housing is a peg 48 which serves as a meansfor aligning the slide by engaging the notch 3 (see FIG. 4a) in thefront end of the slide. Also there is a depression 49 on a slide supportmember 50 which further aligns the slide so that the porous filtermembrane 5 (see FIG. 4a) is located above the photoresponsive electrode45 and also the predetermined areas in the porous filter membrane 51where analytes or analyte complexes are located are aligned above thetransparent circular spots 46 on the photoresponsive electrode. Thehousing also defines a liquid chamber 52 and there is an exit 53 and anentrance port 54 for filling and emptying to liquid chamber 52. Theliquid in the liquid chamber is in contact with the photoresponsiveelectrode and the porous filter membrane. The liquid serves as anelectrolyte and contains a reagent that reacts with the analyte oranalyte complex on the porous filter membrane. For example, if theanalyte complex contains an enzyme, the liquid in the liquid chamberwill contain an enzyme substrate and this enzyme/enzyme substratereaction causes a local change in pH on the surface of thephotoresponsive electrode. As shown in FIG. 7 the slide reading assemblyhas a plunger 60 resiliently mounted by flexing member 55 in the top ofthe housing. The plunger 60 has a stem 61 and a pivotally mounted pad62. The ball and socket mounting 65 gives flexibility to the mounted pad62.

FIG. 7 shows light from LED 47 passing through holes 43 in the innerwall 44 and through the transparent spots 46 on the photoresponsiveelectrode 45 which are aligned with the predetermined areas 51 in theporous filter membrane 5 where analytes or analyte complexes arelocated.

This pivotally mounted pad 62 is pushed against the porous filtermembrane 5 by a stepping motor 63 which is operatively associated withthe stem 61 of the plunger 60. This pad 62 reduces the volume of liquidabove the porous filter membrane and greatly increases the sensitivityof the photoresponsive electrode. FIG. 7a illustrates the holes 43 inthe inner wall section 44.

U.S. Ser. No. 065,418 filed Jun. 18, 1987 and assigned to the sameassignee as this application describes in great detail thephotoresponsive electrode and the increase in sensitivity resulting fromreducing the volume of liquid above the membrane. The specification ofU.S. Ser. No. 065,418 is incorporated herein by reference. The presentinvention differs from the invention described in U.S. Ser. No. 065,418in that the slide is aligned so that spots on the membrane havinganalyte or analyte complex are aligned above the irradiated portion ofthe photoresponsive electrode and the pivotally mounted pad uniformlycompresses the areas of the porous filter membrane where the analyte islocated to exclude excess volume of buffering electrolyte so as thegreatly increase sensitivity of the photoresponsive electrode formeasuring potential changes resulting from the reaction of enzymesubstrate and enzyme bound to the porous filter membrane due to thepresence of analyte or analyte complex.

The operation of the photoresponsive electrode, including the referenceelectrode controlling electrode, materials for the electrode, lightsource, nature of measurable chemical reactions, signal amplificationand measurement, types of measurable enzyme and redox reactions and thelike are described in great detail in U.S. Pat. Nos. 4,704,353;4,758,786 and 4,591,550, which are incorporated here by reference. Aschematic of an electrical circuit usable in the present invention isshown in U.S. Pat. No. 4,758,786.

FIG. 8 shows a preferred circuit for use with this invention. Shown inFIG. 8 is a schematic diagram of a computer-controlled electroniccircuit which may be used to operate the potentiometric reading devicein accordance with the present invention. A photoresponsive electrode 45(e.g. n-type 10-25 ohm-cm, 100 crystalline silicon) polished on one sideis covered on the polished side with an insulator 82 which is in contactwith an electrolyte 84 enclosed by a chamber wall 86 sealed to theinsulator surface by a silicon polymer 88. Operational amplifiers 90 and92, together with resistors 94 and 96, reference electrode 98,controlling electrode 100, and digital-to-analog converter (D/A) 118mounted on master circuit board 102, function to determine the potentialof the electrolyte with respect to the bulk of the photoresponsiveelectrode 45. Computer 112, having keyboard 114, varies the potential ofelectrolyte 84 via the output of D/A 118. The potential of thephotoresponsive electrode 45 is held constant at virtual ground bycopper lead 106 which is attached to the underside of thephotoresponsive electrode 45 through a brass spring and ohmic contact104. Ohmic contact 104 is made by evaporation of 99% gold--1% arseniconto the non-insulated silicon surface followed by alloying above thegold-silicon eutectic temperature. One light-emitting diode (LED) 47 ofan array of nine similar LEDs (not shown) is powered by LED driver 110so as to irradiate the photoresponsive electrode at the desiredX-Y-coordinate with light of 50% duty cycle, on/off-modulated intensity.Any one of the nine similar LEDs may be selected for light intensitymodulation by computer 112 which acts on a switching circuit in LEDdriver 110. The frequency of intensity-modulation is determined by LEDdriver 110 to be about 10 KHz. Current-to-voltage converter 107,connected through lead 106, measures the alternating photocurrentgenerated in photoresponsive electrode 45. The voltage output ofcurrent-to-voltage converter 107, is filtered by bandpass filter 109 andrectified by rectifier 111 to give a dc voltage output that isproportional to the alternating current amplitude. This analog dcvoltage output is converted into digital form by analog-to-digitalconverter (A/D) 116 and stored as data in the memory of computer 112.Experimentally-acquired data may be viewed on CRT display 120 andpermanently displayed by printer 122.

In operation, modulated current is applied from LED driver 110 to causeLED 47 to be modulated in intensity. The output of D/A 118 is ramped bya program in computer 112 so as to ramp the potential of electrolyte 84.The electrolyte potential, in turn, affects the electrical field withinthe photoresponsive electrode which, in turn, affects the alternatingcurrent generated in the illuminated portion of photoresponsiveelectrode 45, flowing through lead 106, and measured bycurrent-to-voltage converter 107. FIG. 9 shows a typical alternatingcurrent response to changes in the electrolyte potential is determined.The values of current vs. electrolyte potential are stored forsubsequent analysis of the rate of change in this relationship. In apreferred embodiment of this invention, the point of maximum slope inthe curve of FIG. 9 is determined, i.e. that point at which theresulting alternating photocurrent finds its maximum changes for a givenchange in electrolyte potential. A convenient way of determining thispoint of maximum slope of the photocurrent of FIG. 9 is to take thesecond derivative and determine where the second derivative is equal tozero. This is depicted in the graph of FIG. 9a. To avoid considerationof the data points where the alternating photocurrent is varying onlyslightly vs. electrolyte potential, the date near the maximum or minimumalternating photocurrents are not used in the analysis. For example, thedata points associated with photocurrent less than 10% and more than 90%of the maximum alternating photocurrent are neglected. Alternatively,the same objective may be achieved by considering only data pointsbetween the largest maximum and smallest minimum of the secondderivative of curve in FIG. 9. Alternative electronic circuits, methodsof analysis, and variety of materials that may be employed inphotoresponsive potentiometric reading devices are disclosed in U.S.patent application, "Photoresponsive Detection and Discrimination," U.S.Ser. No. 231,091, filed Aug. 11, 1988, assigned to the same assignee asthis application and herein incorporated by reference.

The measured rate of change in the relationship between the alternatingphotocurrent amplitude and the electrolyte potential is determined bythe rate of change of the electrostatic surface potential present at theelectrolyte-exposed surface of the photoresponsive electrode at the X-Ycoordinate of irradiation with intensity-modulated light (Science 240,1182-1185, May 27, 1988). This surface potential is determined by the pHof the electrolyte exposed to the surface when a pH-sensitive surface,e.g. silicon nitride is employed and is redox potential sensitive whenthe surface is a noble metal, e.g. platinum or gold (see U.S. Ser. No.231,981, 072,168, and 065,418).

A photoresponsive electrode can be influenced by the redox potential ofthe medium adjacent the electrode surface. Various redox systems can beemployed, enzyme reactions, particularly oxidoreductases, e.g., glucoseoxidase, peroxidase, uricase, NAD or NADP dependent dehydrogenases,naturally occurring electron transfer agents, e.g., ferridoxin,ferritin, cytochrome C, and cytochrome b₂, organic electron donors andacceptor agents, e.g., methylene blue, nitro tetrazolium, Meldola blue,phenazine methosulfate, metallocenes, e.g., ferrocenium, naphthoquinone,N,N'-dimethyl 4,4'-dipyridyl, etc., and inorganic redox agents, e.g.,ferri- and ferroxyanide, chloronium ion, cuprous and cupric ammoniumhalide, etc.

According to the foregoing specification, the slide reading assembly hasa photoresponsive electrode with a multiplicity of measurement sites,and includes both a reference and a controlling electrode, as shown inFIG. 8. Alternatively, the reference electrode may be made optional byshorting the output of operation amplifier 90 and the input ofoperational amplifier 92, shown in FIG. 8, and making a commonconnection to a single counter electrode placed in the electrolyte 84.The counter electrode may be extremely simply, namely a foil or wire ofa metal such as platinum, gold, iridium, titatium, etc. which is inertto the electrolyte. In this alternative mode, the photoresponsiveelectrode has a multiplicity of measurement sites that includes at leastone control site where no analyte is present and is thereby able to, bydifference, measure the rate of potential change generated by thechemical reaction due to presence of the analyte at other sites at thesurface of the photoresponsive electrode.

In operation the stepper motor 63 in FIG. 7 is engaged with the plungerstem 61 to push the pad 62 of the plunger against the porous filtermembrane. A chemical reaction occurs at the surface of thephotoresponsive electrode between reagent in the liquid such as anenzyme substrate with an enzyme which is part of an analyte complex.Light from an LED 47 goes through the holes 43 in the housing and isabsorbed within the photoresponsive electrode 45. The chemical reactionsuch as the reaction of urease with urea to release ammonia and carbondioxide causes a change in pH or redox potential of the electrolyte.This in turn affects the surface potential of the photoresponsiveelectrode, which is turn alters the effect of light on thephotoresponsive electrode.

DNA Assay Standard Curve

Membrane: biotin-BSA coated nitrocellulose membrane (0.8μ pore side).

DNA sample: 0, 5, 10, 25, 50, 100, 150, 200 pg of single-stranded Calfthymus DNA in 0.5 ml of phosphate buffered saline buffer 50 mm NaPO₄,150 NaCl, 1 mm EDTA; 0.05% sodium azide pH 7.0.

Reagent: 0.5 μg/ml Streptavidin, 1 ng/ml SSB-biotin, 250 ng/mlanti-DNA-urease, 0.1% BSA in 10 mM tris·HCl buffer, 1 mM EDTA (pH 7.4)plus 0.25% triton X-100, 0.05% sodium azide.

Assay Protocol: 500 μl of DNA sample was incubated with 1000 μl ofreagent at 37° for 60 minutes. The mixture was filtered through thebiotin-BSA coated membrane at a flow rate of about 100 μl/min. Themembrane was then washed with 1 cc of wash buffer (10 mm NaPO₄, 100 mmNaCl, 0.05% Tween 20, 0.05% sodium azide, pH 6.5) at a maximum flow rateof about 6 ml/min). After washing, the slide was transferred to theslide reading assembly where the liquid contains as 100 mM urea in thewash as substrate.

    ______________________________________                                        Results                                                                       DNA (Pg/sample)                                                                              Results of Signal (μV/Sec)                                  ______________________________________                                        0              42.0                                                           5              75.5                                                           10             91.0                                                           25             177.5                                                          50             331                                                            100            656                                                            150            938                                                            200            1353                                                           ______________________________________                                    

The results are shown as a curve in FIG. 10. Thus the analytical workstation permits the determination of the picogram (pg) qualities of DNAat quantities as low as 2 pg over a dynamic range of 2-200 pg.

What is claimed is:
 1. A slide reading assembly comprising:(a) a housingwith a top, bottom, and inner and outer sidewall sections wherein theinner side wall section has a window with a photoresponsive electrodehaving an inner surface mounted above the window on the inside of theinner side wall section so that light from an external source can passinto and be absorbed within the photoresponsive electrode and said outersidewall section has a surface;the top section of the housing having anopening for receiving a slide to be analyzed wherein the slide has aporous filter membrane with analyte to be determined on predeterminedlocations on the porous filter membrane, said housing having means foraligning the predetermined locations on the porous filter membrane onthe inner surface of the photoresponsive electrode opposite the windowin the housing which allow light to pass to a photoresponsive electrode;a resilient plunger mounted in the outer side wall surface of thehousing opposite the photoresponsive electrode, the plunger having astem and pad such that when the plunger stem is pushed inwardly, by anoperatively associated stepping motor, into the housing the padcompresses the porous membrane against the inner surface of thephotoresponsive electrode; a liquid chamber within the housing with exitand entrance ports for liquids in the top section of the housing andwherein the inner surface of the photoresponsive electrode is within theliquid chamber and the porous filter membrane is aligned above the innersurface of the photoresponsive electrode in the liquid chamber; (b) anintensity-modulated light source for independently irradiating thephotoresponsive electrode through the window in the housing on each areaof the photoresponsive electrode opposite the area of the porous filterhaving a predetermined location of filtered analyte or analyte complex;and (c) an electrical means for measuring a potential change of thesurface of the photoresponsive electrode when the photoresponsiveelectrode is exposed to an intensity-modulated beam of light through thewindow in the housing and a chemical reaction is occurring between thefiltered analyte or analyte complex on the porous filter membrane and areagent in the liquid in the liquid chamber, the electrical meansincluding a counter electrode, and an operation amplifier.
 2. A slidereading assembly according to claim 1 wherein the inner surface of thephotoresponsive electrode has a coating of insulation.
 3. A slidereading assembly according to claim 1 wherein the pad of the plunger ispivotally mounted on the stem of the plunger.
 4. A slide readingassembly according to claim 1 wherein the slide alignment means is aslide support member with a depression that fits the configuration ofthe slide and a peg that fits a notch in the slide.
 5. A slide readingassembly according to claim 1 wherein the alignment means is a peg atthe bottom section of the housing and a depression on a slide supportmember to fit the shape of the slide.
 6. A slide reading assemblyaccording to claim 1 which has a reference electrode.
 7. A slide readingassembly according to claim 5, said slide comprising a thin flatgenerally rectangular plastic sheet with a front and rear end whereinthe front end of the slide has a notch as means for aligning the slideboth in a filter assembly and a reading assembly; the rear end of theslide having an area for manually grasping the slide; wherein the front,approximately a third of the slide, defines an opening which is coveredwith a porous filter membrane and a section of the front one third ofthe slide is narrower than the rear section of the slide, said narrowersection also serving as a means for aligning the slide in both thefiltering assembly and the reading assembly and wherein the alignmentmeans provide for deposition of analyte complex onto predeterminedlocations on the porous filter membrane in the filter assembly and fordetection of analyte complex at these same predetermined locations inthe reading assembly.
 8. A slide reading assembly according to claim 6wherein the porous filter membrane has fixed to it a hapten and whereinthe hapten is biotin.
 9. A slide reading assembly according to claim 5which has a buffering capacity of 1-10 m mole per liter of compressedmembrane volume.
 10. An analytical work station comprising one or morefiltering assemblies and a slide reading assembly, said filteringassembly comprising in combination:a block with a top and bottomsurface, said block having a plurality of channels with inlet ports onthe top surface and exit ports on the bottom surface and a firstalignment means; a vacuum plate having an inner and an outer surfacewherein the outer surface has a vacuum fitting defining an area on theouter surface, with a plurality of vacuum ports from the inner to outersurface within the area defined by the vacuum fitting, said vacuum platehaving a second alignment means; a slide defining an opening which iscovered by a porous filter membrane and further having a third alignmentmeans; wherein the block and the vacuum plate have spacing meansproviding space for removably inserting the slide between the block andthe vacuum plate and wherein the first, second, and third alignmentmeans align the block, vacuum plate, and slide such that the exit portsof the block, porous filter membrane, and vacuum ports are aligned sothat when a vacuum is applied through the vacuum fitting, liquids in theblock channels separately flow through the exit ports, through theporous filter membrane, and through the vacuum ports without liquidsfrom one channel mixing with liquids from other channels on the porousfilter membrane and wherein analyte or analyte complex in the filteredliquid are fixed on the porous filter membrane in a predeterminedlocation, said slide reading assembly comprising: a housing with a top,bottom and inner and outer side wall sections wherein the inner sidewall section has a window with a photoresponsive electrode mounted abovethe window on the inside of the inner side wall section so that lightfrom an external source can pass into and be absorbed within thephotoresponsive electrode; the top section of the housing having anopening for receiving a slide to be analyzed wherein the slide has aporous filter membrane with analyte to be determined on predeterminedlocations on the porous filter membrane, said housing having means foraligning the predetermined locations on the porous filter membrane onthe inner surface of the photoresponsive electrode opposite the windowin the housing which allow light to pass to a photoresponsive electrode;a resilient plunger mounted in the outer side wall surface of thehousing opposite the photoresponsive electrode, the plunger having astem pad such that when the plunger stem is pushed inwardly, by anoperatively associated stepping motor, into the housing the padcompresses the porous membrane against the inner surface of thephotoresponsive electrode; a liquid chamber within the housing with exitand entrance ports for liquids in the top section of the housing andwherein the inner surface of the photoresponsive electrode is within theliquid chamber and the porous filter membrane is aligned above the innersurface of the photoresponsive electrode in the liquid chamber; anintensity-modulated light source for independently irradiating thephotoresponsive electrode through the window in the housing on each areaof the photoresponsive electrode opposite the area of the porous filterhaving a predetermined location of filtered that or that complex; and anelectrical means for measuring the potential change of the surface ofthe photoresponsive electrode when the photoresponsive electrode isexposed to an intensity-modulated beam of light through the window inthe housing and a chemical reaction is occurring between the filteredthat or that complex on the porous filter membrane and a reagent in theliquid in the liquid chamber, the electrical means including a counterelectrode, and an operational amplifier.
 11. In combination, a filteringassembly and a slide reading assembly, said filtering assemblycomprising in combination:a block with a top and bottom surface, saidblock having a plurality of channels with inlet ports on the top surfaceand exit ports on the bottom surface and a first alignment means; avacuum plate having an inner and outer surface wherein the outer surfacehas a vacuum fitting defining an area on the outer surface, with aplurality of vacuum ports from the inner to outer surface within thearea defined by the vacuum fitting, said vacuum plate having a secondalignment means; a slide having a front end and sides, the slidedefining an opening which is covered by a porous filter membrane andfurther having a third alignment means; wherein the block and the vacuumplate have spacing means providing space for removably inserting theslide between the block and the vacuum plate and wherein the first,second, and third alignment means align the block, vacuum plate, andslide such that the exit ports of the block, porous filter membrane, andvacuum ports are aligned so that when a vacuum is applied through thevacuum fitting, liquids in the block channels separately flow throughthe exit ports, through the porous filter membrane, and through thevacuum ports without liquids from one channel mixing with liquids fromother channels on the porous filter membrane and wherein analyte oranalyte complex in the filtered liquid filtered through the membrane arefixed on the porous filter membrane in predetermined locations, saidthird alignment means providing additionally for positioning the slidein the slide reading assembly so that detection will occur at the samepredetermined locations on the porous membrane, and said slide readingassembly comprising a housing with a top, bottom and inner and outerside wall sections, said inner side wall section having an electrodemounted thereon, the top section of the housing having an opening forreceiving the slide and the housing having a fourth alignment means foraligning the predetermined locations on the porous membrane tocorresponding predetermined locations on the surface of the electrode.12. A filter assembly according to claim 11, wherein the first alignmentmeans is a hole in the bottom surface of the block; the second alignmentmeans is a peg in the vacuum plate that is received by the hole; thethird alignment means is a notch in the front end of the slide, whichengages the peg; and the top surface of the vacuum plate has adepression that conforms to the shape of the sides of the slide.
 13. Incombination, one or more filtering assemblies, each comprising a blockwith a top and bottom surface, said block having a plurality of channelswith inlet ports on the top surface and exit ports on the bottom surfaceand a fist alignment means;a vacuum plate having an inner and outersurface wherein the outer surface has a vacuum fitting defining an areaon the outer surface, with a plurality of vacuum ports from the inner toouter surface within the area defined by the vacuum fitting, said vacuumplate having a second alignment means; a slide having a front end andsides, the slide defining an opening which is covered by a porous filtermembrane and further having a third alignment means; wherein the blockand the vacuum plate have spacing means providing space for removablyinserting the slide between the block and the vacuum plate and whereinthe first, second, and third alignment means align the block, vacuumplate, and slide such that the exit ports of the block, porous filtermembrane and vacuum ports are aligned so that when a vacuum is appliedthrough the vacuum fitting, liquids in the block channels separatelyflow through the exit ports, through the porous filter membrane, andthrough the vacuum ports without liquids from one channel mixing withliquids from other channels on the porous filter membrane and whereinanalyte or analyte complex in the liquid filtered through the membraneare fixed on the porous filter membrane in predetermined locations; aslide reading assembly cooperating with the filtering assembly, saidslide reading assembly comprising a housing with a top, bottom and innerand outer side walls, said inner side wall having an electrode mountedthereon, the housing including a top section having an opening forreceiving the slide, said slide being positioned so that detection willoccur at the same predetermined locations on the porous membrane, andsaid housing having a fourth alignment means for aligning thepredetermined locations on the porous membrane to correspondingpredetermined locations on the surface of the electrode.
 14. Thecombination of claim 13 wherein the third alignment means providesadditionally for positioning the slide in the slide reading assembly sothat detection will occur at the same predetermined locations on theporous membrane.
 15. In combination, a filtering assembly comprising ablock with top and bottom surface, said block having a plurality ofchannels with inlet ports on the top surface and exit ports on thebottom surface and a first alignment means; a vacuum plate having aninner and outer surface wherein the outer surface has a vacuum fittingdefining an area on the outer surface, with a plurality of vacuum portsfrom the inner to outer surface within the area defined by the vacuumfitting, said vacuum plate having a second alignment means; a slidehaving a front end and sides, the slide defining an opening which iscovered by a porous filter membrane and further having a third alignmentmeans; wherein the block and the vacuum plate have spacing meansproviding space for removably inserting the slide between the block andthe vacuum plate and wherein the first, second, and third alignmentmeans align the block, vacuum plate, and slide such that the exit portsare aligned so that when a vacuum is applied through the vacuum fitting,liquids in the block channels separately flow through the exit ports,through the porous filter membrane, and through the vacuum ports withoutliquids from one channel mixing with liquids from other channels on theporous filter membrane and wherein analyte or analyte complex in theliquid filtered through the membrane are fixed on the porous filtermembrane in predetermined locations, and a slide reading assemblycomprising a housing with a top, bottom, and inner and outer sidewallsections wherein the inner side wall section has a window with anelectrode mounted thereon; the housing having a top section having anopening for receiving said slide to be analyzed; and wherein the thirdalignment means provides additionally for positioning the slide in saidslide reading assembly so that detection will occur at the samepredetermined locations on the porous membrane a photoresponsiveelectrode having an inner surface, means mounted in the outer side wallsurface of the housing for compressing the porous membrane against theinner surface of the photoresponsive electrode; a liquid chamber withinthe housing with exit and entrance ports for liquids in the top sectionof the housing and wherein the inner surface of the photoresponsiveelectrode is within the liquid chamber and the porous membrane isaligned above the inner surface of the photoresponsive electrode in theliquid chamber a window on the housing on each side of thephotoresponsive device; an intensity-modulated light source forindependently irradiating the photoresponsive electrode through saidwindow in the housing on each side of the photoresponsive electrodeopposite the area of the porous membrane and an electrical means formeasuring a potential change of the surface of the photoresponsiveelectrode when the photoresponsive electrode is exposed to anintensity-modulated beam of light through the window in the housing anda chemical reaction is occurring between the filtered analyte or analytecomplex on the porous filter membrane and a reagent in the liquid in theliquid chamber.